Sulfonamide aminomethylene derivatives as immunosuppressants

ABSTRACT

Compounds which suppress human T-lymphocyte proliferation are disclosed. The active compounds essentially contain at least the following structure: ##STR1##

This application is a continuation of application Ser. No. 08/015,502filed on Feb. 9, 1993, now abandoned.

FIELD OF THE INVENTION

The invention relates to methods and compounds for controllinginflammatory processes in humans through mediation of inflammatory cellproliferation. More particularly, the present invention is a method forsuppressing T-lymphocytes using a class of novel compounds.

BACKGROUND

Compounds which retard the production of cytokines such as interleukin-2(IL-2) are known. For instance, U.S. Pat. No. 4,764,503 assigned toSandoz Ltd., Basel, Switzerland, describes a compound genericallyreferred to as Cyclosporin A (hereinafter referred to as "CsA"), andU.S. Pat. No. 4,894,366 assigned to Fujisawa Pharmaceuticals, Osaka,Japan, describes a compound they designate as "FKS06." Both CsA and FK506 are claimed to inhibit IL-2 production and bind to cellular receptorproteins that possess Peptidyl Prolyl Isomerase (PPIase) activity(Johansson et al., 1990, Transplantation 50:10017).

It was initially postulated by those skilled in the art that thespecific binding by such compounds to PPIase type proteins led toinhibition of the protein's isomerase activity which, in turn, led toinhibition of T-cell proliferation. Thus, these PPIase type proteinswere referred to as "immunophilins", with the cellular receptor proteinsthat bound to CsA and FK506 being referred to as "cyclophilin" and"FKS06 binding protein", respectively. FK506 binding protein is alsosimply referred to as "FKBP" (Harding et al., 1989, Nature 341:758).

Recent publications report that the inhibition of PPIase activity, inand of itself, is not sufficient for immunosuppressant activity.However, there is support in the literature that inhibitory binding toPPIase-type enzymes probably contributes to ultimate T-cell suppression(Sigal et al. 1991, J. Exp. Med. 173:619).

This disclosure presents a new class of synthetic compounds that bothsuppress the proliferation of T-cells and inhibit the isomerase activityof the FKBP-type of PPIases.

CsA, a cyclic undecapeptide, has received FDA approval for use as anadjunct to organ transplant procedures. However, CsA is administeredwith caution due to its known toxicity. Currently, CsA is prescribed insituations where the risks of non treatment outweigh the risks of itstherapeutic complications.

As a result, efforts to expand the application of CsA into non lifethreatening indications such as chronic maintenance of autoimmunedisorders have been limited by the well-known side effects of this drug.The use of CsA leads to a variety of disorders including:nephrotoxicity, such as impairment of glomerular filtration andirreversible interstitial fibrosis (Kopp et al., 1991, J. Am. Soc.Nephrol. 1:162 ); neurological deficits, such as involuntary tremors, ornon-specific cerebral angina such as non-localized headaches (De Groenet al, 1987, N. Engl. J. Med. 317:861); and vascular hypertension withcomplications resulting therefrom (Kahan et al., 1989, N. Engl. J. Med.321:1725).

Recent efforts to investigate the cause of the adverse effects of CsAadministration have centered on the role of CsA breakdown into toxicmetabolites (Bowers et al., 1990, Clin. Chem. 36:1875; Burke et al.,1990, Transplantation 50:901). The prevailing thought is that CsAtoxicity is due to such metabolites and not due to the nature of the CsAbinding to the PPIase, cyclophilin (Akagi et al., 1991, J. Int. Med.Res. 19:1; Ryffel et al., 1988, Transplantation 46:905).

Thus, inhibitor compounds that do not resemble CsA structurally, yetbind to PPIases, should be more amenable to therapeutic applications.Such non-toxic immunosuppressors would benefit the art, especially forchronic administration such as required in the treatment of autoimmunedisorders.

The compound FK506 is structurally different from CsA and does notproduce the same type of toxic metabolites. FK506 has been shown to beeffective in some transplant patients who do not respond to CsA (Tucciet al., 1989, J. Immunol. 143:718).

However, testing of FK506 in humans was delayed due to severe vasculitisobserved in treatment regimens in dogs and baboons (Collier et al.,1988, Transplant Proc. 20:226). Furthermore, other clinical side effectsand complications of FK506 administration are being reported (Frayha etal., 1991, Lancet 337:296; Kitahara et al., 1991, Lancet 337:1234). Ithas also been reported that "overall, the absolute rate of clinicalrejection in FK506 [post-organ transplantation] patients is onlyslightly lower than with current standard therapies" (Holechek, 1991,Anna. J. 18:199).

In an attempt to alleviate the FK506 side effects, many minormodifications to the base structure have been reported. For example,U.S. Pat. No. 5,057,608 assigned to Merck & Co. and WIPO Publication No.W089/05304 assigned to FISONS PLC Inc. both disclose chemical variationsof the FKS06 compound.

To date only a few studies on the metabolism of FK506 have beenpublished, and little information has been reported on the toxicity ofits metabolites (Johansson et al., 1990, Transplantation 50:1001;Christians et al., 1991, Clinical Biochemistry 24:271; Lhoest et al.,1991, Pharmaceutica Acta Helveticae 66:302). Since it is likely that thepatterns of metabolism of the FK506 analogs and derivatives are similarto the parent compound, it is also likely that many of the side effectsof FK506 will be shared by the derivatives.

As is true for CsA, the toxicity of FK506 is postulated to be based onits structure and not due to its binding activity with the immunophilinFKBP. It is further postulated that the toxicities of compounds such asCsA and FK506 are due to various chemical groups found in thesestructures which do not participate in the immunosuppressive activity,such as those groups which result in the toxic metabolites of CsAbio-processing. Thus, relatively compact molecules which do not resembleeither CsA or FK506, and which have both immuno-suppressive and PPIasebinding activity should be free of side effects associated with CsA andFK506.

The present invention presents a novel class of synthetic inhibitorcompounds. The novel class includes synthetic aminomethylene derivativesthat bind to human FKBP-type PPIases and demonstrate human peripheralT-lymphocyte inhibitory activity.

Amino-methylene derivatives are known. For example, several claimedamino-methylene HIV inhibitors have been published, including WO90/00399 assigned to Smithkline Beckman Corp., EP 0387231 assigned toWashington University, and EP 0361341 assigned to Miles Inc., byassignment from Molecular Therapeutics, Inc. Similarily, amino-methyleneinhibitors of the enzyme renin have also been published, including EP0374097 assigned to CIBA Geigy AG. Also published are amino methylenecompounds which are claimed to be therapeutics for neurologicdysfunctions such as EP 374,756 assigned to Merck Inc.

As used herein, the term "aminomethylene-prolyl spacer" refers to apeptide fragment in which the carbonyl of the central amide bond hasbeen replaced by an alkyl fragment such as a methylene group. ##STR2##

It is therefore an object of the present invention to provide forcompounds and compositions containing such aminomethylene derivativesfor suppression of pathological and abnormal human peripheralT-lymphocyte proliferation.

It is also an object of the present invention to provide a novel classof compounds suitable for therapeutic compositions designed to suppresspathological immune responses, such as the hyperimmune response in organtransplantation rejection, the self-destructive autoimmune diseases, andthe overproduction and excessive proliferation of immune cells such asin infectious disease states.

More specific objects include provisions for compounds, compositions andmethods for treatment and prevention of rejection of transplanted organsor tissues such as kidney, heart, lung, liver, bone marrow, skin grafts,and corneal replacement.

It is a further object to provide compounds, compositions and methodsfor use in the treatment of autoimmune, degenerative, inflammatory,proliferative and hyperproliferative diseases, such as rheumatoidarthritis, osteoarthritis, other degenerative joint diseases, jointinflammation such as associated with infectious diseases such assuppurative arthritis, and secondary arthritis such as those associatedwith gout, hemochromatosis, rheumatic fever, Sjorgens syndrome andtuberculosis.

Another object is to provide compounds, compositions and methods for usein the treatment of lupus erythematosus, systemic lupus erythematosus,Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type 1diabetes, uveitis, nephrotic syndrome, and of cutaneous manifestationsof immunologically-mediated diseases such as psoriasis, atopicdermatitis, contact dermatitis, eczematous dermatitides, seborrheicdermatitis, lichen planus, pemphigus, bollous pemphigoid, epidermolysisbullosa, urticaria, angioedemas, vasculitides, erythemas, cutaneouseosinophilias, and alopecia areata.

Yet another object is to provide compounds, compositions and methods foruse in the treatment of abnormal T-cell proliferation such aslymphocytic leukemia; Hodgkin's disease, especially those subtypesinvolving abnormal T-cell subpopulations; non-Hodgkin's lymphomas, suchas mycosis fungoides, convulated lymphocytic lymphoma, and immunoblasticsarcoma; and chronic lymphadenitis.

The above lists are non-limiting, and one skilled in the art couldeasily adapt the compounds, compositions and methods of the presentinvention to other indications, such adaptations being within the spiritand scope of the invention which will be described hereinbelow.

SUMMARY OF THE INVENTION

The presently claimed invention relates to active compounds of thefollowing generalized structure: ##STR3## where R⁰ is straight orbranched alkyl (C1-C10), cycloalkyl (C3-C10), bicycloalkyl (C5-C12),tricycloalkyl (C7-C14), or tetracycloalkyl (C9-C16). These alkylderivatives may be substituted by a doubly bonded oxygen atom in such away as to form a ketone. These alkyl derivatives may also be substitutedby straight or branched alkoxy (C1-C3), cyano, carboalkoxy (withstraight or branched alkoxy of C1-C6), alkylsulfonyl (C1-C5), or up tothree times by fluoro. R⁰ may also be an quinolyl, styryl, or aryl.Throughout this application, aryl refers to phenyl derivatives such asphenyl, napthyl, and related aromatic, carbon derivatives. These arylderivatives may be substituted up to three fold by straight or branchedalkyl (C1-C6). R⁰ may also be heteroaryl (six membered with up to 2nitrogens), or a 5-membered ring heterocycle such as furan, thiophene,1,3-thiazole; these heterocycles may be substituted by an acetylamino ora five membered ring heterocycle containing nitrogen or oxygen or both.

R¹ is hydrogen or methyl.

R² and R³ are defined as follows: one of R² and R³ is hydrogen and theother is straight or branched alkyl (C1-C6) that may be substituted bycycloalkyl (C6), hydroxy, straight or branched alkoxy (C1-C4),benzyloxy, or aryl. The aryl may be substituted by hydroxy or methoxy.

n=an integer of 1, 2 or 3.

X¹ is oxygen or NR⁴, where R⁴ is hydrogen or methyl.

J is the divalent fragment ##STR4## where R⁵ is hydrogen, straight orbranched alkyl (C1-C8). These alkyl derivatives could be substituted byhydroxy, straight or branched alkoxy (C1-C6), benzyloxy, cycloalkyl(C6), or phenyl.

K is one of the fragments ##STR5## --HC═CH-- or --(CH₂)_(p) --where R⁶is hydrogen or methyl and where it is understood that the carbonyl groupis attached to "J" and that NR⁶ is connected to "L", respectively,

p=an integer of 1, 2, 3 or 4.

L is hydrogen, aryl, straight or branched alkyl (C1-C8) that may besubstituted by aryl. The aryl group may be substituted by alkoxy(C1-C5), hydroxy, or amino. These aminoaryl derivatives may besubstituted by straight or branched alkyl (C1-C6) or acetyl or both.

Included within the scope of the present invention are pharmacueticallyacceptable salts of the above mentioned compounds. Pharmaceuticallyacceptable salts can be derived from mineral acids, carboxylic acids orsulfur containing acids, preferably from hydrochloric acid, hydrobromicacid, sulfuric acid, methane sulfonic acid, ethane sulfonic acid,toluene sulfonic acid, benzene sulfonic acid, naphthalene disulfonicacid, acetic acid, propionic acid, lactic acid, tartaric acid, citricacid, fumaric acid, maleic acid or benzoic acid. Most preferred are thehydrochlorides.

In the case of the present compounds being carboxylic acids orcontaining acidic functional groups, the invention includes metal saltsand ammonium salts. Preferred are sodium, potassium or ammonium salts.The compounds of this invention exist as stereoisomeric forms, whicheither behave like image and mirror image (enantiomers) or not(diastereomers). Included within the scope of the invention are theenantiomers, the racemic forms and diastereomeric mixtures. Enantiomersas well as diastereomers can be separated by methods known to thoseskilled in the art (See E. L. Eliel, Stereochemistry of CarbonCompounds, McGraw Hill, 1962).

The presently claimed invention also relates to active compounds of thefollowing generalized structure: ##STR6## where R⁰ is straight orbranched alkyl (C1-C8), cycloalkyl (C3-C8), bicycloalkyl (C5 -C10),tricycloalkyl (C7-C 12 ), or tetracycloalkyl (C9-C14). These alkylderivatives may be substituted by a doubly bonded oxygen atom in such away as to form a ketone. These alkyl derivatives may also be substitutedby fluoro, methoxy, cyano, carboalkoxy (with straight or branched alkoxyof C1-C4), or an alkylsulfonyl (C1-C4). R⁰ may also be an 8-quinolyl,styryl, aryl, or aryl that may be substituted up to three fold bystraight or branched alkyl (C1-C6). R⁰ may also be heteroaryl (sixmembered with up to 2 nitrogens), or a 5-membered ring heterocycle suchas furan, thiophene, 1,3-thiazole; these heterocycles may be substitutedby an acetylamino or a five membered ring heterocycle containingnitrogen or oxygen or both.

R¹ is hydrogen or methyl.

R² and R³ are defined as follows: one of R² and R³ is hydrogen and theother is straight or branched alkyl (C1-C6) that may be substituted bycycloalkyl (C6) or phenyl.

n=an integer of 1, 2 or 3.

X¹ is oxygen or NR⁴, where R⁴ is hydrogen or methyl.

J is the divalent fragment ##STR7## where R⁵ is hydrogen, straight orbranched alkyl (C1-C6). These derivatives could be substituted byhydroxy, alkoxy (C1-C6), benzyloxy, cycloalkyl (C6), or phenyl.

K is one of the fragments ##STR8## --HC═CH-- or --(CH₂)_(p) --where R⁶is hydrogen or methyl and where it is understood that the carbonyl groupis attached to "J" and that NR⁶ is connected to "L", respectively,

p=an integer of 2 or 3.

L is hydrogen, aryl, straight or branched alkyl (C1-C6) that may besubstituted by aryl. The aryl group may be substituted by alkoxy(C1-C5), hydroxy, or amino. These aminoaryl derivatives may besubstituted by alkyl (C1-C4) or acetyl or both.

Included within the scope of the present invention are pharmacueticallyacceptable salts of the above mentioned compounds. Pharmaceuticallyacceptable salts can be derived from mineral acids, carboxylic acids orsulfur-containing acids, preferably from hydrochloric acid, hydrobromicacid, sulfuric acid, methane sulfonic acid, ethane sulfonic acid,toluene sulfonic acid, benzene sulfonic acid, naphthalene disulfonicacid, acetic acid, propionic acid, lactic acid, tartaric acid, citricacid, fumaric acid, maleic acid or benzoic acid. Most preferred are thehydrochlorides.

Preferred compounds of the present invention have the followinggeneralized structure: ##STR9## where R⁰ is straight or branched alkyl(C1-C6). These alkyl derivatives may be substituted by a methylsulfonyl.R⁰ may also be styryl or a tricycloalkyl of C10, substituted by a doublybonded oxygen atom in such a way as to form a ketone. R⁰ may also be an8-quinolyl, aryl, or aryl that may be substituted up to three fold bystraight or branched alkyl (C1-C3). R⁰ may also be heteroaryl (sixmembered with 1 nitrogen), or a 5-membered ring heterocycle such asfuran, thiophene, 1,3-thiazole; these heterocycles may be substituted byan acetylamino or an isoxazol-3-yl.

R¹ is hydrogen.

R² and R³ are defined as follows: one of R² and R³ is hydrogen and theother is straight or branched alkyl (C1-C4) that may be substituted bycycloalkyl (C6).

n=an integer of 1, 2 or 3.

X¹ is oxygen or NR⁴, where R⁴ is hydrogen.

J is the divalent fragment ##STR10## where R⁵ is hydrogen, straight orbranched alkyl (C1-C4). K is one of the fragments ##STR11## --HC═CH-- or--(CH₂)_(p) --where R⁶ is hydrogen and where it is understood that thecarbonyl group is attached to "J" and that NR⁶ is connected to "L",respectively.

p=an integer of 2.

L is hydrogen, phenyl, straight or branched alkyl (C1-C3) that may besubstituted by aryl.

Included within the scope of the present invention are pharmacueticallyacceptable salts of the above mentioned compounds. Most preferred arethe hydrochlorides.

Preferred Method of Synthesis

Synthesis of Dipeptide Derivatives ##STR12##

Imino acid derivatives could be dehydratively coupled to N-protected,amino acid derivatives using standard coupling agents such as PPA, DCCor other reagents as described in standard books on peptide coupling(such as Bodanszky et al. The Practice of Peptide Synthesis:Springer-Verlag, Vol 21, 1984). The group used to protect the nitrogenof these amino acids could be either carbotertbutoxy, carbobenzyloxy,carboallyloxy, or other temporary protecting groups as described in theliterature (T. W. Greene et al, Protective Groups in Organic Synthesis,2nd Edition; John Wiley & Sons, 1991 ).

Synthesis of Aminomethylenes ##STR13##

A convenient route to prepare the present compounds involves reductionof the central peptide bond as depicted in equation 2.0. Standardmethods to effect transformations of this type have been reported(Cushman, M. et al. J. Org. Chem. 1991, 56, 4161-7). For example, theintermediate amide bond can be reduced with a borohydride reagent suchas borane in a polar solvent such as tetrahydrofuran, ether, ordimethoxyethane.

Alternatively, these derivatives may also be prepared by a procedure inwhich the amide bond is first converted to a thioamide intermediateusing sulfur transfer reagents such as Lawesson's Reagent (Synthesis1979, 941). The resulting thioamide obtained by this or other proceduresmay be reduced to the corresponding aminomethylene derivative bytreatment with a reducing reagent such as Raney nickel. In cases wherethe thioamide derivative is a phenylmethyl ester derivative, reductionof the thioamide may lead directly to the corresponding reducedderivative containing a free carboxylic acid group (Eq. 2.1) ##STR14##

Formation of Aminomethylene Carboxylic Acids. ##STR15##

Although methods as depicted in Eq. 2.1 may allow for the formation ofcarboxylic acid derivatives directly from the corresponding thioamidephenylmethyl esters, these intermedates are also obtained from thecorresponding esters (Eq. 3). Conditions used to effect hydrolysis orconversion of ester derivatives to acid derivatives are described indetail in the literature (T. W. Greene et al, Protective Groups inOrganic Synthesis, 2nd Edition; John Wiley & Sons, 1991).

C-Terminal Homologation of Aminomethylenes ##STR16##

Aminomethylene-carboxylic acid derivatives prepared as shown in Eq. 2.1,Eq. 3.0, or by other methods can be dehydratively coupled to a varietyof alcohol or amines to provide the corresponding ester (X═O) or amidederivatives (X═NR). This dehydrative coupling can be achieved withstandard coupling agents such as PPA, DCC or other reagents as describedin standard books on peptide coupling (such as Bodanszky The Practice ofPeptide Synthesis: Springer-Verlag, Vol 21, 1984).

Deprotection of Aminomethylene N-Termini ##STR17##

The deprotection of amine derivatives has been described (T. W. Greeneet al, Protective Groups in Organic Synthesis, 2nd Edition; John Wiley &Sons, 1991). These methods are useful for the conversion of protectedamine derivatives to free amino derivatives.

Formation of Sulfonamides ##STR18##

Amine derivatives prepared using methods illustrated by Eq. 5.0 or fromother sources may be treated with sulfonyl halides in halogenatedsolvents such as dichloromethane, 1,2-dichloroethane or chloroform toform the corresponding sulfonamides.

Alternatively, sulfonamide derivatives may be prepared in openingN-protected aziridine derivatives as shown in Eq. 7.0. ##STR19##

The presently claimed compounds were found to be effective at lowmicromolar doses in both in vivo assays for inhibition ofmitogen-induced human T-cell proliferation and NF-AT directedβ-galactosidase expression. Moreover, the results from the rat adjuvantarthritis model (described in detail further below) indicate that thepresent class of compounds exhibits desirable biological properties(prophylactic prevention of paw swelling), at the concentration tested(10 mg/kg/dose).

The present invention encompasses pharmaceutical formulations which, inaddition to non-toxic, inert pharmaceutically suitable excipients,contain the compounds of the invention.

The present invention also includes pharmaceutical formulations indosage units. This means that the formulations are present in the formof individual part, for example, tablets, dragees, capsules, caplets,pills, suppositories and ampules, the active compound content of whichcorresponds to a fraction or a multiple of an individual dose. Thedosage units can contain, for example, 1, 2, 3 or 4 individual doses; or1/2, 1/3 or 1/4 of an individual dose. An individual dose preferablycontains the amount of active compound which is given in oneadministration and which usually corresponds to a whole, one half, onethird or one quarter of a daily dose.

By non-toxic inert pharmaceutically suitable excipients there are to beunderstood solid, semi-solid or liquid diluents, fillers and formulationauxiliaries of all types.

Preferred pharmaceutical formulations which may be mentioned aretablets, dragees, capsules, caplets, pills, granules, suppositories,solutions, suspensions and emulsions, paste, ointments, glues, creams,lotions, dusting powders and sprays. Tablets, dragees, capsules,caplets, pills and granules can contain the active compounds in additionto the customary excipients, such as (a) fillers and extenders, forexample, starches, lactose, sucrose, glucose, mannitol and silicic acid,(b) binders, for example, carboxymethylcellulose, alginates, gelatin andpolyvinylpyrrolidone, (c) humectants, for example, glycerol, (d)disintegrating agents, for example, agar-agar, calcium carbonate andsodium carbonate, (e) solution retarders, for example, paraffin and (f)absorption accelerators, for example, quaternary ammonium compounds, (g)wetting agents, for example, cetyl alcohol and glycerol monostearate,(h) absorbents, for example, kaolin and bentonitc and (i) lubricants,for example, talc, calcium stearate, magnesium stearate and solidpolyethylene glycols, or mixtures of the substances listed under (a) to(i) directly hereinabove.

The tablets, dragees, capsules, caplets, pills and granules can beprovided with the customary coatings and shells, optionally containingopacifying agents and can also be of such composition that they releasethe active compounds only or preferentially in a certain part of theintestinal tract, optionally in a delayed manner. Examples of embeddingcompositions which can be used are polymeric substances and waxes.

The active compounds can also be present in microencapsulated form, ifappropriate with one or more of the abovementioned excipients.Suppositories can contain, in addition to the active compounds, thecustomary water-soluble or water-insoluble excipients, for example,polyethylene glycols, fats, for example, cacao fat and higher esters(for example, C 14 -alcohol with C16 -fatty acid), or mixtures of thesesubstances.

Ointments, pastes, creams and gels can contain, in addition to theactive compounds, the customary excipients, for example, animal andvegetable fats, waxes, paraffins, starch tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures of these substances.

Dusting powders and sprays can contain, in addition to the activecompounds, the customary excipients, for example, lactose, talc, silicicacid, aluminum hydroxide, calcium silicate and polyamide powder, ormixtures of these substances. Sprays can additionally contain customarypropellants, for example, chlorofluorohydrocarbons.

Solutions and emulsions can contain, in addition to the activecompounds, customary excipients, such as solvents, solubilizing agentsand emulsifiers, for example, water, ethyl alcohol, isopropyl alcohol,ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,propylene glycol, 1,3-butylene glycol, dimethylformamide, oils, inparticular, cottonseed oil, groundnut oil, corn germ oil, olive oil,castor oil and sesame oil, glycerol, glycerol formal, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, ormixtures of these substances.

For parenteral administration, the solutions and emulsions can also bein a sterile form which is isotonic with blood. Suspensions can contain,in addition to the active compounds, customary excipients, such asliquid diluents, for example, water, ethyl alcohol or propylene glycoland suspending agents, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum methydroxide, bentonitc, agar-agar, and tragacanth,or mixtures of these substances.

The abovementioned pharmaceutical formulations can also contain otherpharmaceutical active compounds in addition to the claimed compounds ofthe present invention.

The aforementioned pharmaceutical formulations are prepared in thecustomary manner by known methods, for example, by mixing the activecompound or compounds with the excipient or excipients.

The formulations mentioned can be used either with humans and animals,orally, rectally, bucally, parenterally (intra-venously, intramuscularlyor subcutaneously), intracisternally, intravaginally, intraperitoneallyor locally (dusting powder, ointment or drops) and for the therapy ofinfection in hollow spaces or body cavities. Suitable formulations areinjection solutions, solutions and suspensions for oral therapy, gels,pour-on formulations, emulsions, ointments or drops. Ophthalmologicaland dermatological formulations, silver salts and other salts, eardrops, eye ointments, powders or solutions can be used for localtherapy.

It is furthermore possible to use gels, powders, dusting powders,tablets, sustained release tablets, premixes, concentrates, granules,pellets, capsules, caplets, aerosols, sprays and inhalates on humans andanimals. The compounds according to the invention can furthermore beincorporated into other carrier materials, such as, for example,plastics (e.g., chains of plastic for local therapy), collagen or bonecement.

DETAILED DESCRIPTION

The following describes a preferred way to prepare the compounds of thepresent invention.

REAGENTS AND INSTRUMENTS:

Anhydrous tetrahydrofuran (THF), ethyl ether (Et₂ O), and acetonitrilewere distilled from calcium hydride prior to use. Unless otherwisestated, all reagents discussed in the following examples werecommercially available from Aldrich Chemical Co, Milwakee, Wis., orJanssen Chimica through the U.S. vender Spectrum Chemicals Mfg. Corp.,New Brunswick, N.J.

All reactions were carried out in oven-dried glassware (140° C.) whichwere cooled under argon prior to use. Crude products were purified byflash column chromatography using 230-400 mesh silica gel (35-70 um) ormedium/high pressure liquid chromatography using Shimadzu LC-8APreparative liquid chromatography system equipped with columns packedwith either 20 um or 10 um silica. Thin layer chromatography (TLC) wasperformed on aluminum-backed silica gel plates, and visualization wasaccomplished with a UV light or an iodine vapor chamber.

Proton (¹ H) nuclear magnetic resonance (NMR) spectra were obtained on aGN-OMEGA-300 spectrometer at 300 MHz. Carbon (¹³ C) NMR were obtained onthe same spectrometer at 75 MHz. Mass spectral data were obtained on aKratos-MS 80RFA spectrometer using electron impact ionization (El),chemical ionization (CI), or fast atom bombardment (FAB). Other massspectral (MS) data were obtained on a Kratos CONCEPT I-H spectrometer,using liquid-cesium secondary ion (LSI) technique, a more modern versionof fast atom bombardment (FAB).

Melting points were obtained on a Thomas Hoover capillary melting pointapparatus in open-ended capillaries and are not corrected.

Abbreviations used in the following experimental section refer to thefollowing reagents: DCC is 1,3-dicyclohexyl carbodiimide; DMAP is4-dimethylaminopyridine; TFA is trifluoroacetic acid; HOBT is1-hydroxybenzotriazole monohydrate.

Amino acid derivatives described as 1-[X]-L-Isoleucine are meant tosignify a derivative of the the L-isomer of the amino acid Isoleucine,in which the α-amino group is attached to the the fragment X. In asimiliar fashion, 1-[1-[X]-L-Proline]-L-Isoleucine is meant to representa fragment that can be represented graphically as: ##STR20##

Example 1 ##STR21## 1-[2-(S)-Methanesulfonylamino-4-methylpentyl]-L-proline4-Phenylbutylamide.

a) 1-Thio-1-[2-(S)-[[(1,1-dimethylethoxy)carbonyl]amino]-4-methylpentyl]-L-Proline Benzyl Ester.This compound was prepared from in 49% yield using the proceduredescribed earlier (Synthesis, 1979, 941). The ¹ H NMR of this compoundwas consistent with the structure. Rf=0.61 (2% methanol indichloromethane).

b)1-[2-(S)-[[(1,1-dimethylethoxy)carbonyl]amino]-4-methylpentyl]-L-Proline.Thetitle compound of Example 1a (8.14 g) was dissolved in absolute ethanol(30 mL) and treated with #2-Raney nickel (60 mL 1:1 v/v slurry inabsolute ethanol) at 22° C. for 2 hours. The reaction was filtered on aglass frit and washed with ethanol (700 mL). The filtrate wasconcentrated under reduced pressure and chromatographed on silica gel toprovide 1.05 g (18%) of the title compound and 835 mg (11%) of the1-[2-(S)-[[(1,1-dimethylethoxy)carbonyl]amino]-4-methylpentyl]-L-ProlineBenzyl ester. The ¹ H NMR of both these compounds were consistent withtheir structures.

c)1-[2-(S)-[[(1,1-Dimethylethoxy)carbonyl]amino]-4-methylpentyl]-L-proline4-phenylbutylamide. The title compound of Example 1b (392 mg),1,3-dicyclohexylcarbodiimide (386 mg), dimethylaminopyridine (152 mg),1-hydroxybenzotriazole monohydrate (191 mg) and triethylamine (350 uL)were dissolved in N,N-dimethylformamide (1 mL) and dichloromethane (2mL) and were treated with 4-phenylbutylamine (296 uL). The reaction wasstirred for 12 hours at 20° C., then concentrated under reducedpressure. The residue was dissolved in dichloromethane and washed withsatd aq NaHCO₃, satd aq NaCl, dried (MgSO₄), and concentrated underreduced pressure to an oil. The oil was chromatographed on acidic silicagel to provide 356 mg (64%) of the title compound. The ¹ H NMR and Massspectrum of this compound were consistent with the structure. Rf=0.26(50% EtOAc in hexane). LSIMS=446; (mass calculated for C₂₆ H₄₃ N₃ O₃=445.62).

d) 1-[2-(S)-Amino-4-methylpentyl]-L-proline 4-Phenylbutylamide. Thetitle compound of Example 1c (455 mg) was dissolved in dichloromethane(3 mL) and treated with trifluoroacetic acid (3 mL). The reaction wasstirred at 20° C. for 3 hours, then concentrated under reduced pressure.The residue was dissolved in dichloromethane and was washed with said aqNaHCO₃, said aq NACl, dried (MgSO₄), and concentrated under reducedpressure to provide 332 mg (94%) of the free amine. The ¹ H NMR of thiscompound was consistent with the structure, and the amine was useddirectly for subsequent reactions.

1e) 1-[2-(S)-Methanesulfonylamino-4-methylpentyl]-L-proline4-Phenylbutylamide. In a round bottom flask were added1-[2-(S)-amino-4-methylpentyl]-L-proline 4-phenylbutylamide (20 mg),triethylamine (20 uL) and anhydrous dichloromethane (1 mL). The solutionwas cooled to -78° C., and methane sulfonyl chloride (7 uL) was added.After the addition was complete, the solution was allowed to warm to 0°C. and then to 22° C. The reaction mixture was washed with said aqNaHCO₃, said aq NaCl, dried (MgSO₄), evaporated under reduced pressure,and chromatographed on silica to provide 11 mg (45%) of the titlecompound. The ¹ H NMR and Mass spectrum of this compound were consistentwith the structure. Rf=0.38 (EtOAc). LSIMS=424; (mass calculated for C₂₂H₃₇ N₃ O₃ S=423.60).

Example 2 ##STR22##1-[2-(S)-(Toluene-4-sulfonylamino)-4-methylpentyl]-L-proline4-Phenylbutylamide. Using the procedure described in Example 1e,treatment of 1-[2-(S)-amino-4-methylpentyl]-L-proline 4-phenylbutylamide(20 mg) with p-toluenesulfonyl chloride (16 mg) provided 13 mg (44%) ofthe title compound. The ¹ H NMR and Mass spectrum of this compound wereconsistent with the structure. Rf=0.54 (EtOAc). LSIMS=500; (masscalculated for C₂₈ H₄₁ N₃ O₃ S=499.69). Example 3 ##STR23##1-[2-(S)-(Quinoline-8-sulfonylamino)-4-methylpentyl]-L-proline4-Phenylbutylamide. Using the procedure described in Example 1e,treatment of 1-[2-(S)-amino-4-methylpentyl]-L-proline 4-phenylbutylamide(40 mg) with 8-quinolinesulfonyl chloride (40 mg) provided 33 mg (54%)of the title compound. The ¹ H NMR and Mass spectrum of this compoundwere consistent with the structure. Rf=0.44 (EtOAc). LSIMS=537; (masscalculated for C₃₀ H₄₀ N₄ O₃ S=536.71). Example 4 ##STR24##1-[2-(S)-(2-Acetylamino-4-methyl-S-thiazolesulfonylamino)-4-methylpentyl]-L-proline4-Phenylbutylamide. Using the procedure described in Example 1e,treatment of 1-[2-(S)-amino-4-methylpentyl]-L-proline 4-phenylbutylamide(116 mg) with 2-acetamido-4-methyl-5-thiazolsulfonyl chloride (116 mg)provided 60 mg (35%) of the title compound. The ¹ H NMR and Massspectrum analysis of this compound were consistent with the structure.Rf=0.49 (4.8% methanol in dichloromethane). LSIMS=564; (mass calculatedfor C₂₇ H₄₁ N₅ O₄ S₂ =563.76). Example 5 ##STR25##1-[2-(S)-(Thiophene-2-sulfonylamino)-4-methylpentyl]-L-proline4-Phenylbutylamide. Using the procedure described in Example 1e,treatment of 1-[2-(S)-amino-4-methylpentyl]-L-proline 4-phenylbutylamide(115 mg) and 2-thiophenesulfonyl chloride (80 uL) provided 107 mg (75%)of the title compound. The ¹ H NMR and Mass spectrum of this compoundwere consistent with the structure. Rf=0.49 (4.8% methanol indichloromethane). LSIMS=492; (mass calculated for C₂₅ H₃₇ N₃ O₃ S₂=491.69). Example 6 ##STR26##1-[2-(S)-(2-phenyl-ethenesulfonylamino)-4-methylpentyl]-L-proline4-Phenylbutylamtde. Using the procedure described in Example 1e,treatment of 1-[2-(S)-amino-4-methylpentyl]-L-proline 4-phenylbutylamide(124 mg) with trans-betastyrenesulfonyl chloride (82 mg) provided 111 mg(67%) of the title compound. The ¹ H NMR and Mass spectrum of thiscompound were consistent with the structure. Rf=0.51 (EtOAc). LSIMS=512;(mass calculated for C₂₉ H₄₁ N₃ O₃ S=511.70). Example 7 ##STR27##1-[2-(S)-(7,7-Dimethyl-2-oxo-bicyclo[2.2.1]hept-1-ylmethanesulfonylamino)-4-methylpentyl]-L-proline4-Phenylbutylamide. Using the procedure described in Example 1e,treatment of 1-[2-(S)-amino-4-methylpentyl]-L-proline 4-phenylbutylamide(132 mg) with (-)-10-camphorsulfonyl chloride (108 mg) provided 99 mg(51%) of the title compound. The 1H NMR and Mass spectrum of thiscompound were consistent with the structure. Rf=0.56 (EtOAc). LSIMS=560;(mass calculated for C₃₁ H₄₉ N₃ O₄ S=559.78). Example 8 ##STR28##1-[2-(S)-(5 - [Isoxazol- 25 -yl]-thiophene-2-sulfonylamino)-4-methylpentyl]-L-proline 4-Phenylbutylamide. Using theprocedure described in Example 1e, treatment of1-[2-(S)-amino-4-methylpentyl]-L-proline 4-phenylbutylamide (123 mg)with 5-(isoxazol-3-yl)thiophene-2-sulfonyl chloride (Maybridge Chemical:125 mg) provided 24 mg (13%) of the title compound. The ¹ H NMR and Massspectrum analysis of this compound were consistent with the structure.Rf=0.28 (3.2% methanol in dichloromethane). LSIMS=559; (mass calculatedfor C₂₈ H₃₈ N₄ O₄ S₂ =558.73). Example 9 ##STR29##1-[2-(S)-(Methylsulfonylmethanesulfonylamino)-4-methylpentyl]-L-proline4-Phenylbutylamide. Using the procedure described in Example 1e,treatment of 1-[2-(S)-amino-4-methylpentyl]-L-proline 4-phenylbutylamide(114 mg) with methylsulfonylmethylsulfonyl chloride (Maybridge Chemical:103 mg) provided 38 mg (26%) of the title compound. The ¹ H NMR and Massspectrum of this compound were consistent with the structure. Rf=0.56(EtOAc). LSIMS=502; (mass calculated for C₂₃ H₃₉ N₃ O₅ S₂ =501.68).Example 10 ##STR30##1-[2-(S)-(2',4',6'-Triisopropylbenzenesulfonylamino)-4-methylpentyl]-L-proline4-Phenylbutylamide. Using the procedure described in Example 1e,treatment of 1-[2-(S)-amino-4-methylpentyl]-L-proline 4-phenylbutylamide(136 mg) with 2,4,6-triisopropylbenzenesulfonyl chloride (163 mg)provided 75 mg (34%) of the title compound. The ¹ H NMR and Massspectrum of this compound were consistent with the structure. Rf=0.65(EtOAc). LSIMS=632; (mass calculated for C₃₆ H₅₇ N₃ O₃ S=611.90).Example 11 ##STR31##1-[1-[2-(S)-ethanesulfonylamino-3-(S)-methylpentyl]-L-homoprolyl]-L-isoleucineBenzylamide. Using the procedure described in Example 1e,1-[2-(S)-amino-3-(S)-methylpentyl]-L-homopropyl]-L-isoleucinebenzylamide (722 mg) was acylated at -78° C. with methane sulfonylchloride (130 uL) to give 347 mg (41%) of the title compound. The ¹ HNMR and Mass spectrum of this compound were consistent with thestructure. Rf=0.59 (EtOAc). LSIMS=509; (mass calculated for C₂₆ H₄₄ N₄O₄ S=508.70).

The immunosuppressive properties of the present compounds were evaluatedin the following assays:

1) Inhibition of PPlase Activity

This assay follows in principle the procedure described in Kofron etal., 1991, Biochemistry 30:6127. The three main reagents used arePPIase, a substrate for PPIase, and a selected inhibitor compound of thepresent invention. The basic principle behind this assay is theconversion of the cis isomer of the substrate to the trans form, whichconversion is catalyzed by PPIase. Essentially, inhibition of thisPPIase activity is measured for the selected compounds. A peptidechymotrypsin substrate containing a proline in the P2 position is onlycleaved by chymotrypsin when the Phe-Pro bond is in the trans isomericconfiguration. In the presence of excess chymotrypsin, all of the transpeptide isomers are cleaved within approximately five seconds, leavingonly cis forms.

The cis peptide will spontaneously convert to the trans isomer at a slowrate. The cis to trans conversion is catalyzed by isomerases at a muchfaster rate than this spontaneous conversion. Proteins with PPIaseactivity are examples of such isomerases. After isomerization, thepeptide is cleaved by chymotrypsin releasing p-nitroaniline which can bemonitored at 390 nm. The rate of release is then calculated using afirst order rate plus offset equation utilizing the ENZFITTER program(Leatherbarrow, BIOSOFT, Cambridge, United Kingdom).

Example 12

PPIase Inhibition Assay

In a plastic cuvette are added 950 ul of ice cold assay buffer (25 mMHEPES, pH 7.8, 100 mM NaCl), 10 uL of FKBP (2.5 uM in 10 mM Tris-Cl pH7.5,100 mM NACl, 1 mM dithiothreitol), 25 ul of chymotrypsin (50 mg/mlin 1 mM HCl) and 10 ul of the test compound at various concentrations indimethyl sulphoxide. The reaction is initiated by addition of 5 ul ofsubstrate (Succinyl-Ala-Phe-Pro-Phe-para-nitroanilide, 5 mg/ml in 235 mMLiCl in trifluoroethanol).

The absorbance at 390 nm versus time is monitored for 90 sec using aBeckman DU70 spectrophotometer. The absorbance versus time data filesare transferred to an IBM XT computer and the rate constants determinedusing the commercial Enzfitter program. For each set of data, theuncatalyzed rate of conversion is measured and the uninhibited enzymaticrate determined. The data are expressed as % Inhibition and arecalculated as follows: ##EQU1## where k_(obs) is the rate in thepresence of a selected test compound, k_(uncat) is the rate in theabsence of enzyme, and k_(uninh) is the rate in the presence of enzymeand absence of inhibitor. Data are plotted as percent inhibition versusconcentration of inhibitor. The values of the concentration of inhibitorrequired for 50% inhibition of enzyme activity (IC₅₀) were determined bynonlinear least squares regression analysis.

                  TABLE 1                                                         ______________________________________                                        Example No.   FKBP IC.sub.50 (μM)                                          ______________________________________                                        1             3.5                                                             2             >5                                                              3             >5                                                              4             >5                                                              5             >5                                                              6             >5                                                              7             >5                                                              8             >5                                                              9             >5                                                              10            >5                                                              11            >5                                                              ______________________________________                                    

Results: The results of the compound testing are presented in Table 1,above. As stated previously, it was not initially apparent whether ornot inhibition of PPIase activity was necessary and sufficient forimmunosuppression. Presently, the prevailing thought is that binding tothe PPIase enzyme may be necessary but is not sufficient. Therefore, thedata on PPIase inhibition may be viewed as an assay to detect whether ornot a given compound is capable of interacting productively with FKBP.

2) Human T Lymphocyte Inhibition

Inhibition of mitogen-induced T-cell proliferation can be used toprofile immunosuppressive activity of test compounds. In the descriptionof the assay which follows, mitogen-induced T-cell proliferation wasused to test the inhibitory potencies of select compounds of the presentinvention.

In an assay similar to that described by Bradley in Mishell et al.(Eds.), 1980, Selected Methods in Cellular Immunology, pp 156-161, W. H.Freeman & Co., San Fransisco, Calif., T-cells were stimulated byincubation with phytohemagglutinin (PPIA) which binds to cell surfacemolecules, including the T-cell receptor. This stimulation results inproliferation which can be measured by incorporation of [³ H]-thymidineinto cellular DNA.

The immunosuppressive properties of the compounds of the presentinvention can be determined by adding various concentrations of thecompounds to these cultures and measuring the effect on T-cellproliferation.

Example 13

Suppression of Human T-Cell Proliferation Assay

Fresh LeukoPaks were obtained from the New York Blood Center, New York,N.Y. The cells, including erythrocytes and leukocytes, were diluted withHank's Balanced Salt Solution (HBSS) (GIBCO, Grand Island, N.Y.) andlayered over Lymphoprep (Nycomed Pharma AS, Oslo, Norway) in sterile 50ml conical centrifuge tubes. Lymphocytes were isolated at theHank's/Nycomed interface after centrifugation at 2000×g, 4° C. for 15min. The lymphocytes were washed with Minimal Essential Medium (GIBCO)containing 2% fetal bovine serum (FBS) (Sigma Chemical Co., St. Louis,Mo.), 1% HEPES buffer (GIBCO) and 1% Penicillin-Stretomycin solution(GIBCO).

T-cells were further purified essentially by sheep erythrocyte (SRBC)rosetting as described by Morimoto et al., 1983, J. Immunol. 130:157.The isolated lymphocytes were adjusted to 2×10⁷ cells/ml and 5 mlaliquots of the cell suspension were incubated for 10 minutes at roomtemperature with 5 ml of a 5% SRBC (Cappel, Organon Technika Corp., WestChester, Pa.) suspension. The cells were gently pelleted bycentrifugation at 300 rpm for 10 minutes, followed by a 1 hourincubation at room temperature to allow rosette formation. The cellswere gently resuspended, layered over Lymphoprep and centrifuged for 30minutes at 500×g. The pellet, containing rosetted T-cells and SRBC wastreated with ice cold buffered ammonium chloride (GIBCO) to lyse theerythrocytes. T-cells were washed twice with HBSS.

Purified T-cells were resuspended at 2×10⁶ cells/ml in complete culturemedium composed of RPMI-1640 (Whittaker Bioproducts, Walkerville, Md.)with 10% FBS (Sigma), 2 mM L-glutamine (GIBCO), 1%Penicillin-Streptomycin (GIBCO) and 15 mM HEPES (GIBCO). In 96-wellplates (Becton Dickinson, Lincoln Park, N.J.), 0.1 ml aliquots of T-cellsuspension were mixed with 0.05 ml of 40 μg/ml PPIA-M (Sigma). Thecompounds of this invention were dissolved in dimethylsulfoxide at 10 mMand various dilutions in complete medium were added in duplicate wells(0.05 ml/well). The plates were incubated at 37° C. in a humidifiedatmosphere of 5% carbon dioxide and 95% air for 72 hours.

Proliferation was assessed by measurement of [3H]-thymidineincorporation. During the last 6 hours of incubation, the cells werepulse labelled with 1 μCi/well of [3H]-thymidine (New England Nuclear,Boston, Mass.). The cells were harvested onto glass fiber paper using aplate harvester and the radioactivity incorporated into cellular DNAcorresponding to individual wells was measured by standard liquidscintillation counting methods. The mean counts per minute (CPM) ofreplicate wells was calculated and linear regression analysis of meanCPM versus compound concentration was used to determine theconcentration of compound which would inhibit [³ H]-thymidineincorporation of T-cells by 50% (IC₅₀).

The results of this assay, presented in Table 2, are representative ofthe intrinsic immunosuppresive activity of the compounds of the presentinvention. Thus, concentrations less than 10 μM of some of the preferredcompounds suppress the T-cell proliferative response by 50%.

                  TABLE 2                                                         ______________________________________                                        Example No.    IC.sub.50 (μM)                                              ______________________________________                                        1              2                                                              2              8                                                              3              5                                                              4              2                                                              5              3                                                              6              5                                                              7              2                                                              8              1                                                              9              4                                                              10             4                                                              11             >15                                                            ______________________________________                                    

3) NF-AT Assay

Stimulation of T-cells leads to the appearance of several transcriptionfactors, including one designated "NF-AT". These factors are involved inregulation of gene expression required for immunologic activation. Someof these transcription factors appear to have functions in a widevariety of cell types. By contrast, NF-AT is found primarily in T-cellsand its role is restricted to early gene activation. In addition, NF-ATactivity is inhibited by the immunosuppressant drugs, Cyclosporin A andFK506 (Schreiber and Crabtree, 1992, Immunology Today 13:136).

Inhibition of NF-AT activity is measured using FGL-5 cells. FGL-5 is acloned line of stably transfected Jurkat T-cells that contain aconstruct in which three tandem copies of the NF-AT DNA binding sitedirect transcription of the lacZ gene, encoding β-galactosidase (Fieringet al., 1990, Genes & Development 4:1823). When these cells arestimulated with phorbol esters which activate protein kinase C andcalcium ionophore to raise the intracellular calcium concentration,transcriptionally active NF-AT is produced. In T-cells, this normallyleads to the expression of IL-2, T-cell growth factor. However, in FGL-5cells NF-AT activation leads to the production of β-galactosidase whichcan be detected using an appropriate substrate.

FGL-5 cells were cultured with phorbol ester, calcium ionophore and thecompounds of the present invention to measure inhibition ofβ-galactosidase activity, as shown below.

Example 14

NF-AT Inhibition Assay Directed β-Galactosidase Expression

This assay was performed essentially as described (Bierer et al., 1990,Proc. Natl. Acad. Sci. 87:9231). FGL-5 cells were maintained in mediumconsisting of RPMI-1640 with 10% FBS, 2 mM L-glutamine, 1% Penicillin-Streptomycin and 15 mM HEPES buffer. The assays were done withexponentially growing cells whose density was not greater than 0.5million cells/ml. The cells were resuspended to 3 million cells/ml inmedium and 0. 1 mi was added to wells of a 96-well plate.

The compounds of the present invention were dissolved in either ethanolor dimethylsulfoxide at 10 mM and 0.05 ml/well of various dilutions inmedium were added to cells in duplicate wells. Treatment controlsconsisted of duplicate wells to which 0.05 ml/well of either medium,ethanol or dimethylsulfoxide was added. The ethanol and dimethylsulfoxide were at the same concentration as was used for the compounds.Cells were incubated with compounds at room temperature for 10-15minutes. Phorbol dibutyrate (Sigma) and Ionomycin (Calbiochem) weredissolved at 50 μg g/mi and 2 mM, respectively and stored at -70° C.

FGL-5 cells were stimulated by diluting these reagents with medium to200 ng/ml and 8 μM, respectively and adding of 0.05 ml/well. Forunstimulated cell controls, 0.05 mi/well of medium was added toduplicate wells. The plates were incubated overnight (16-18 hours) at37° C. in a humidified atmosphere of 5% CO₂ and air. β-galactosidaseactivity was measured as the fluorescence generated by the cleavage of4-methyl umbelliferyl-β-D-galactoside (Sigma) at the β-galactoside bond.After overnight incubation, the cells were centrifuged at 500×g for 3minutes in the 96-well plates and washed 3 times with PBS. The cellswere then resuspended in 0. 18 mi/well of reaction medium containing 100mM sodium phosphate buffer, pH 7.0, 10 mM potassium chloride, 1 mMmagnesium sulfate, 0.1% Triton X-100 (Pierce, Rockford, Ill.), and 0.5mM 4-methylumbelliferyl-β-D- galactoside.

The fluorescence at 460 nm using 355 nm excitation was measured atintervals over 1-2 hours (during which fluorescence increased linearlywith time) with a LS50 Luminescence Spectrometer (Perkin Elmer).

The percent inhibition by each concentration of the compounds wascalculated as: ##EQU2##

The values of the concentration of compounds required for 50% inhibition(IC50) were determined by linear regression analysis of the percentinhibition at various compound concentrations.

The results of this assay presented in Table 3 are representative of theintrinsic immunosuppresive activity of the compounds of the presentinvention. Compounds that inhibited NF-AT directed β-galactosidaseexpression by stimulated FGL-5 cells with IC₅₀ of 11 μM or less alsoinhibited mitogen induced T-cell proliferation, e.g., compounds ofExample Nos. 1 and 3.

                  TABLE 3                                                         ______________________________________                                        Example No.    IC.sub.50 (μM)                                              ______________________________________                                        1              9                                                              2              27                                                             3              11                                                             4              24                                                             5              24                                                             6              >15                                                            7              >15                                                            8              >15                                                            9              ND                                                             10             >15                                                            11             >15                                                            ______________________________________                                         where ND means "not determined                                           

4) Adjuvant Arthritis

Rats sensitized to mycobacterial antigens in Complete Freund's Adjuvantcan develop a rapidly destructive adjuvant arthritis. Adjuvant arthritisappears to be an autoimmune disease. Thus, T lymphocytes from immunizeddonors can transfer the disease to naive recipients (Pearson and Wood,1964, J. Exp. Med. 120:547.) and susceptibility is controlled, at leastin part, by class II MHC genes (Batisto, et al. 1982, Arthritis Rheum.25:1194). The induction of adjuvant arthritis can be inhibited byimmunosuppressant drugs, e.g., Cyclosporin A (Borel, et al., 1976,Agents and Actions. 6:468) and azaspiranes (Badger, et al. 1989, Int. J.Immunopharmac. 11:839)

Example 15

Adjuvant Arthritis Model in the Rat

Complete Freund's adjuvant is made by supplementing extra heavy mineraloil with 10 mg/ml heat killed Mycobacterium butyricum (DifcoLaboratories, Detroit, Mich.). Lewis rats (Charles Rivers, Willmington,Mass.) are given a 0. 1 ml injection of adjuvant (1 mg/animalmycobacterium) subcutaneously into the right hind footpad. In theinjected foot, an acute inflammatory reaction occurs which ischaracterized by erythema, edema and a predominantly neutrophilic cellinfiltrate. This is followed by edema in the uninjected contralateralfoot by days 10-12. This secondary response is accompanied by apredominantly mononuclear cell infiltrate, indicating the presence ofcell-mediated immunity.

The immune response is quantitated by measuring the change in anklediameter of the uninjected hind paw from day 0 to day 16 postsensitization. This is accomplished using a hand-held dial micrometer.Animals are administered test drugs, suspended in a vehicle consistingof 5% polyethylene glycol and 0.5% Tween-80 (Sigma Chemical Co., St.Louis, Mi,) in phosphate buffered saline (GIBCO, Grand Island, N.Y.),i.p. on days -1, 0, 2, S, 7, 9, 12 and 14. Several compounds whenadministered at 10 mg/kg/dose inhibited the swelling in the uninjectedlimb compared with the control groups that were sensitized with CompleteFreund's Adjuvant but received only the vehicle i.p. (Table 4).

                  TABLE 4                                                         ______________________________________                                        Compound    Δ Ankle Diameter                                                                      % Inhibition                                        ______________________________________                                        None        3.3 ± 0.5 mm                                                                             0                                                   Example 1   1.6 ± 0.6 mm                                                                             51                                                  ______________________________________                                    

What is claimed is:
 1. A compound having the structure: ##STR32## whereR⁰ is straight or branched (C₁ -C₁₀)alkyl, (C₃ -C₁₀)cycloalkyl, (C₅-C₁₂)bicycloalkyl, or (C₇ -C₁₄)tricycloalkyl, each of which isoptionally substituted with a straight or branched (C₁ -C₃)alkoxy,cyano, straight or branched carbo(C₁ -C₆)alkoxy, (C₁ -C₅)alkylsulfonyl,one to three fluoro groups, or with a doubly bonded oxygen atom to forma ketone; or R⁰ is styryl, or a phenyl group optionally substituted upto three times with a straight or branched (C₁ -C₆)alkyl;R¹ is hydrogenor methyl; one of R² and R³ is hydrogen and the other is straight orbranched (C₁ -C₆)alkyl optionally substituted with C₆ cycloalkyl,hydroxy, straight or branched (C₁ -C₄)alkoxy, benzyloxy, or aryloptionally substituted with hydroxy or methoxy; n is the integer 2; X¹is NR⁴, where R⁴ is hydrogen or methyl; J is the divalent fragment##STR33## where R⁵ is hydrogen, or straight or branched (C₁ -C₈)alkyloptionally substituted with hydroxy, straight or branched (C₁-C₆)alkoxy, benzyloxy, C₆ cycloalkyl or phenyl; K is one of thefragments --HC═CH-- or --(CH₂)_(p) --where; p is the integer 1, 2, 3 or4; and L is hydrogen, phenyl, or straight or branched (C₁ -C₈)alkyloptionally substituted with phenyl optionally substituted by straight orbranched (C₁ -C₅)alkoxy, hydroxy, or amino, which amino is optionallysubstituted by a straight or branched (C₁ -C₆)alkyl, acetyl, or both. 2.A compound having the structure: ##STR34## where R⁰ is straight orbranched (C₁ -C₈)alkyl, (C₃ -C₈)cycloalkyl, (C₅ -C₁₀)bicycloalkyl, or(C₇ -C₁₂)tricycloalkyl, each of which is optionally substituted with afluoro, methoxy, cyano, straight or branched carbo(C₁ -C₄)alkoxy, (C₁-C₄)alkylsulfonyl or with doubly bonded oxygen to form a ketone; or R⁰is styryl, or a phenyl group optionally substituted up to three times bystraight or branched (C₁ -C₆)alkyl;R¹ is hydrogen or methyl; one of R²and R³ is hydrogen and the other is straight or branched (C₁ -C₆) alkyloptionally substituted by C₆ cycloalkyl or phenyl; n is the integer 2;X¹ is NR⁴, where R⁴ is hydrogen or methyl; J is the divalent fragment##STR35## where R⁵ is hydrogen or straight or branched (C₁ -C₆)alkyloptionally substituted with hydroxy, straight or branched (C₁-C₆)alkoxy, benzyloxy, C₆ cycloalkyl, or phenyl; K is one of thefragments --HC═CH-- or --(CH₂)_(p) --where p is the integer 2 or 3: andL is hydrogen, phenyl, or straight or branched (C₁ -C₆)alkyl optionallysubstituted with phenyl optionally substituted with (C₁ -C₅)alkoxy,hydroxy, or amino, which amino is optionally substituted by (C₁-C₄)alkyl, or acetyl, or both.
 3. A compound having the structure:##STR36## where R⁰ is straight or branched (C₁ -C₆)alkyl optionallysubstituted with methylsulfonyl; orR⁰ is styryl or a C₁₀ tricycloalkylsubstituted by a doubly bonded oxygen to form a ketone; or R⁰ is phenyloptionally substituted up to three times by straight or branched (C₁-C₃)alkyl; R¹ is hydrogen; one of R² and R³ is hydrogen and the other isstraight or branched (C₁ -C₄)alkyl optionally substituted by C₆cycloalkyl; n is the integer 2; X¹ is NH; J is the divalent fragment##STR37## where R⁵ is hydrogen or straight or branched (C₁ -C₄)alkyl; Kis one of the fragments --HC═CH-- or --(CH₂)_(p) --where p is theinteger 2; and L is hydrogen, phenyl, or straight or branched (C₁-C₃)alkyl optionally substituted by aryl.
 4. A therapeutic compositionfor suppressing the proliferation of human T-lymphocytes, comprising aneffective amount of a compound according to claim
 1. 5. A therapeuticcomposition for suppressing the proliferation of human T-lymphocytes,comprising an effective amount of a compound according to claim
 2. 6. Atherapeutic composition for suppressing the proliferation of humanT-lymphocytes, comprising an effective amount of a compound according toclaim
 3. 7. The compound of claim 3 named1-[2-(S)-Methanesulfonylamino-4-methylpentyl]-L-proline4-Phenylbutylamide.
 8. The compound of claim 3 named1-[2-(S)-Toluene-4-sulfonylamino)-4-methylpentyl]-L-proline4-Phenylbutylamide.
 9. The compound of claim 3 named1-[2-(S)-(2-phenyl-ethenesulfonylamino)-4-methylpentyl]-L-proline4-Phenylbutylamide.
 10. The compound of claim 3 named1-[2-(S)-(7,7-Dimethyl-2-oxobicyclo[2.2.1]hept-1-ylmethanesulfonylamino)-4-methylpentyl]-L-proline4-Phenylbutylamide.
 11. The compound of claim 3 named1-[2-(S)-(Methylsulfonylmethanesulfonylamino)-4-methylpentyl]-L-proline4-Phenylbutylamide.
 12. The compound of claim 3 named1-[2-(S)-(2',4',6'-Triisopropylbenzenesulfonylamino)-4-methylpentyl]-L-proline4-Phenylbutylamide.